Pyrococcus furiosus: large-scale cultivation and enzyme purification.

Publisher Summary Pyrococcus furiosus is currently one of the most studied of the hyperthermophilic microorganisms mainly because of the relative ease with which it can be cultivated and good growth yields. The organism was originally isolated from a geothermally heated shallow sea vent off the coast of Italy by Stetter and co-workers. It grows at temperatures ranging from 70° to 105° with an optimum at 100° using complex peptide mixtures or sugars, e.g., starch, glycogen, maltose, and cellobiose as carbon and energy sources. Unlike many other heterotrophic hyperthermophiles, significant growth of P. furiosus is not obligately dependent on elemental sulfur (S°), although if it is added to the growth medium it is reduced by the organism to H 2 S. This chapter presents the methods used to grow P. furiosus up to the 600-liter scale and to prepare cell-free extracts for the large-scale purification of proteins. In addition, a summary is provided of the elution patterns of different enzymes when the cell-free extract is applied to an anion-exchange chromatography column.